Zwitteronic-fatty acid compounds having anti-inflammatory properties

ABSTRACT

A composition and method for treating both superficial and subdermal inflammation is taught by treating an inflamed skin area, muscle, or bone joint, with a therapeutically effective amount of a skin-compatible ester of a zwitterionic aminosulphonic acid (ZASA-Ester) of the formula. ##STR1## wherein M is an alkali metal, like sodium, and R is a naturally occurring, straight-chain, saturated or unsaturated, aliphatic acid, the esters of which form fatty acids in nature. The most useful of which are the acetic oleic (cis isomer), linoleic, palmitic, and stearic moieties; they occur naturally as glycerides, i.e., esters of glycerol. Also useful are analogs of HEPES like piperazine-N&#39;-(2-ethane sulfonic acid); N-methylpiperazine-N&#39;-(2-ethane sulfonic acid);, and the piperiodine analog of the piperazine ester. 
     A pharmacologic composition is also provided which comprises at least one amphoteric (Zwitterionic-ester) with a pharmacologically-acceptable topical carrier or base. Other adjunctive pharmaceutical agents may be included to facilitate other than topical administration. 
     Also useful are analogs of HEPES, like Piperazine-N 1  (2-ethanesulfonic acid; N-Methylpiperazine-N 1  (2-ethanesulfonic acid) and the piperidine analog of the piperazine ester.

CROSS REFERENCE TO OTHER FILINGS

This is a regular patent application submitted for an official filingreceipt under 35 U.S. Code Section 111(a). It relates to DisclosureDocument No. 425,759, filed October 6, 1997, titled: A NewZwitterionic-Fatty Acid Compound That Is An Anti-Inflammatory andHealing Agent, and a Method to Synthesize the Product From Sodium Saltof HEPES and a Fatty Acid Salt. This disclosure is also acontinuation-in-part of my copending provisional application Serial No.60/066,918, filed Nov. 28, 1997.

FIELD OF THE INVENTION

This inventions relates to pharmacologically-active, anti-inflammatorycompounds, and more specifically to long aliphatic chain esters ofselected zwitterionic organic compounds derived from naturally occurringtaurine. This invention pertains to novel zwitterionic compounds withpharmacologic activity including, but not limited to, their use as ananti-inflammatory agents.

BACKGROUND OF THE INVENTION

Any inflammation that occurs in the mammalian body is the clinicalresult of a sequence of events known as the arachidonic acid (ARA)cascade. Cell membranes consist of phospholipids, including fatty acids,one of which is ARA. In the inflammation process, the first step is therelease of ARA from the phospholipid. The next step is the conversion ofARA into the specific mediator of inflammation. One pathway is thecyclooxygenase enzyme, and the other is called the lipoxygenase pathway.Cortisone, along with other selected steroidal agents, block bothinflammation pathways by inhibiting ARA release from the phospholipids.

The mode of action of HEPES-ester is thought to be at the level ofleukotrienes B4, but it is also possible that it occurs at higher levelsin the inflammatory cascade, perhaps at the phospholipase A2 (PPLA2).Successful Inhibition of PPLA2 action would arrest the aforedescribedcascade effect from being initiated.

Medical science searches for other biochemicals that lack the recognizedside effects of prolonged steroid-based (hydrocortisone) medications.One known human biochemical, taurine (2-amino ethanesulfonic acid),synthesis of which occurs in the mammalian liver, has demonstratedanti-inflammatory activity when administered centrally, but not whenadministered subcutaneously or interperitoneally. N-substitutedderivatives of taurine include:4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid; C₈ H₁₈ N₂ O₄ S,which derivatives are commonly identified in the technical literature asHepes (Merck Index, 12th edition monograph #4687). HEPES itself isavailable commercially from Angus Chemicals, as the sodium salt or, asthe free acid. The scientific literature reports that intravenousinjection of (14-C) HEPES, or of (3H) taurine, demonstrated rapidclearance, but with a significantly longer half-life compared withtaurine. Mahon et al theorized that the greater anti-inflammatoryeffects of HEPES (sodium salt and the acid), as compared with taurine,may be due to its slower systemic distribution or clearance, in vivo.The prior art suggest that HEPES is a significant agent to reducecellular inflammation and cellular proliferation. However, the safedelivery systems for the HEPES treatment of inflammation remain to beoptimized.

It is thus a principal object of this invention to provide a HEPES-basedcompound, an ester, and a pharmaceutically acceptable formulationincluding the ester, that is adapted for use in topically appliedproducts so as to reduce symptoms of skin inflammation, wherein theparticular etiology of the inflammation does not call for, or require,the use of antibiotics or germicidal compositions. Improved formulationsfor epidermal penetration, on bruises, muscle strains and sprains arealso areas of useful progressive treatment.

It is another object of the invention to complex the HEPES molecule withselected aliphatic acids, such serving as the active ingredient oftopical applications, which permit the HEPES moiety to penetrate theskin and so to better effect its anti-inflammatory nature.

It is a further object of the invention to provide a HEPES-containingactive ingredient that is not limited to the known subcutaneousinjection or IV infusion routes, but may also effective as a topicalformulation.

A still further object of the invention is to provide HEPES esters as acosmetic formulation ingredient, as a co-emulsifier, usable with topicalanalgesics.

Still another object of the invention is in a cosmetic preparation toincorporate an anti-skin ageing active ingredient.

These and other objects and benefits of this invention will becomeapparent from a study of the following specification.

SUMMARY OF THE INVENTION

The present invention relates to a composition and method for thetreatment of inflammatory conditions in mammals, by the topicaladministration of selected Zwitterionic ester compositions, serving assafe and effective substances. Among useful Zwitterionic compounds whichare presently preferred; these include EPES, PIPES, BES, POPSO, and mostpreferably HEPES, when esterified, then alone, or in combination withother therapeutic ingredients. They are employed by applying to anaffected area of the skin, a therapeutically effective amount of atleast one skin compatible, Zwitterionic-Ester having the genericformula: ##STR2## wherein M is an alkali metal and R is a naturallyoccurring, straight-chain, saturated or unsaturated, aliphatic acid, theesters of which do form fatty acids in nature.

Preferably, a Zwitterionic-ester has at least one pKa value at 20° C. inthe range of 6.0-8.3, to permit its use on human skin, i.e., the esterexists mainly in its dipolar form, in the pH range of 6.0-8.3.

The isoelectric (ISO) point is the pH at which the net charge on amolecule in solution is zero. At this pH, amino acids exist almostentirely in the Zwitterionic state, i.e., the positive and negativegroups are equally ionized. A solution of amino acids at the ISO pointexhibits minimum conductivity, osmotic pressures, and viscosity.

Such dipolar molecules contain, for example, hydroxy groups and aminogroups, and also acid groups, like phosphoric, carboxylic, or sulfonicsacid groups and, generally have pKa's in the range of 6.15-8.4.

Preferred aliphatic values for the ester moiety of the HEPES ester aren-butanoic; isobutyric, n-valeric, palmitic, and stearic, behenyl,lauric, myristic, (and their isomers), among the saturated aliphatics;and oleic and linoleic, among the unsaturated aliphatics.

The invention also provides a pharmaceutical composition for applicationto human skin in the treatment of inflammation comprising at least oneof the above Zwitteronic-esters as the active ingredient, together witha pharmacologically acceptable topical carrier or base.

These select esters may be useful in the treatment of arthritis,myositis, insect bites, sunburn, psoriasis, atopic dermatitis, and otherinflammatory processes of muscle, connective tissue, or skin appendages.

DETAILED DESCRIPTION OF THE INVENTION

The effective proportion of the active ester ingredient, by weight ofthe formulation, is in the range of 1 to 20%, preferably 5 to 10%. Inthe most preferred composition, the effective proportion lies in therange of 6 to 8%. Not every compound falling within the generaldefinition given above, is suitable for topical use per the method ofthe invention. Some few will prove to be contraindicated. Nevertheless,the exclusion of ineffective active ingredients is a matter well withinthe competence of the skilled pharmacologist in the conduct of theanti-inflammatory evaluation protocols (to be described) for disclosedHEPES-esters.

The topical formulation base is selected from a wide variety ofcompositions, formulated according to known principals forpharmaceutical purposes. Such compositions include creams, solids,ointments, lotions, and film-forming solutions among others. They may bepresented in boxes, jars, or compressible tubes, both collapsible andnon-collapsible. The solids may be presented as sticks for rubbing ontothe skin. Some of the topical bases may be presented as papers, woven ornon-woven fabric pieces, or pads, all being impregnated withcomposition.

The invention relates to HEPES derivatives which are pharmacologicallyactive as anti-phospholipase and anti-inflammatory compoundsspecifically, wherein the active ingredients are certain long chainesters of selected zwitterionic compounds, based on an N-substitutedtaurine, namely aliphatic esters of HEPES.

The novel compounds may exist as at least one of the following fiveorganic groups: ester, ether, urethane, amide, or urea of all of thefollowing known compounds, and their salt forms. PreferredZwitterionic-esters are prepared from the below listed sulfonic acids.

ACES--N-(2-Acetamido)-2 amino ethane sulfonic acid.

AMPSO--3-[1,1Dimethyl-2 hydroxyethyl amino] -2-hydroxypropane sulfonicacid.

BES--N,N-bis (Hydroxyethyl)-2 aminoethane sulfonic acid.

DIPSO--3-[N,N-bis (hydroxyl ethyl) amino]-2-hydroxypropane sulfonicacid.

CAPS--3-(cyclohexylamino)-1-propane sulfonic acid.

CAPSO--3-(cyclohexylamino)-1-propane sulfonic acid.

CHES--2-(N-cyclohexylamino) ethane sulfonic acid.

HEPPS--N-(2-hydroxy ethyl) piperazine-N'-3-propane sulfonic acid.

HEPES--N-(2-hydroxy ethyl) piperazine-N'-(2-propane sulfonic acid).

HEPPSO--N-(2-hydroxy ethyl) piperazine-N'-2-hydroxypropane sulfonicacid.

MES--2-(N-Morpholino) ethane sulfonic acid.

MOPS--3-(N-Morpholino) propane sulfonic acid.

MOPSO--3-(N-Morpholino)-2 hydroxy propane sulfonic acid.

PIPES--Piperazine-N, N'-bis (2 ethane sulfonic acid).

New Mono PIPES--Piperazine-N'-(2 ethane sulfonic acid).

POPSO--Piperazine-N, N'-bis (2-hydroxy propane sulfonic acid).

TAPS--3-[N-tris-(hydroxy methyl) methyl amine] -2-hydroxy propanesulfonic acid.

TES--N-tris-(hydroxy methyl)-methyl-2-amine ethane sulfonic acid.

EPES--N-ethylpiperazine-N-(2 ethane sulfonic acid).

The precursor compounds listed above are best known in the literature asbiological buffers. Many are commonly used as buffering agents inmammalian cell cultures. Manufacturers of these compounds include AngusChemicals, SIGMA, and British Drug House.

SYNTHESIS OF HEPES ESTERS

Generally, an alkali metal salt of HEPES is catalytically reacted withan alkyl-substituted, either saturated or unsaturated, aliphatic salt,such as methyl oleate, methyl linoleate, methyl palmitate, methylstearate, methyl myristate, and methyl behenate. They are reacted inequi-molecular amounts, carried out either with or without a non-aqueoussolvent, such as acetone, and in a temperature range of 0° C. tovariable ° C., which is between 0C. and the chosen solvent's refluxtemperature. The purification of the crude ester is carried out by meansof crystallization in an organic solvent, dissolved in methanol, andrecrystallized. As to analytical methodology, preparative high pressureliquid chromatography (HPLC) is followed by TLC, HPLC, and nuclearmagnetic resonance (NMR) techniques. Potentiometric titration and bromictitration are done across the double bond to establish identity andpurity. The HPLC device is equipped with a variable length, ultravioletdetector.

FIRST PROTOCOL TO EVALUATE CANDIDATE FORMULATIONS FOR SUPPRESSION OFINDUCED SKIN INFLAMMATIONS

Sodium lauryl sulfate solution 0.5% is applied in a patch to four areason the volar surface forearm. Site one is pretreated with a baseformula, site two is pretreated with the base formula plus 10% HEPESester, while skin sites three and four are left untreated.

After 24 hours, the sites are examined, the erythema and edema on sitesone and two are recorded by photographs. Site one is treated with thebase product, site three is treated with the base product plus 10% HEPESester, and sites two and four are untreated controls. Each treated site(two) is treated four times a day. The observed results are recorded asthe percent reduction of erythema and edema at the HEPES ester-treatedsites over the base formula site and two control treated sites.

GENERAL SYNTHESIS

Active Ingredients for Synthesis Procedure (that have the HEPES-oleatesynthesis)

Preparation of HEPES (C₈ H₁₈ N₂ O₄ S)--Oleate Ester--Compound I ##STR3##

The preparation of Compound I is characterized by catalytically reacting4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, with salts likemethyl oleate, C₁₇ H₃₃ CO₂ CH₃, in equimolar amounts. The reaction canbe carried out in a non-aqueaous solvent. Sodium methoxide is a usefulcatalyst. The reaction temperature may vary between 0°C. and the solventreflux temperature. The purification of the compound is carried out bymeans of crystallization in a solvent such as acetone.

I. THE ESTERS.

Methyl oleate HEPES Na+Na methoxide →HEPES oleate+Methyl alcohol HEPESNa oleate

HEPES ESTERS GENERIC FORMULA ##STR4##

The preparation of Compound I can be carried out by reacting a fattyacid with HEPES (full compound name) or any one of its salt derivatives.One preferred process route is to react HEPES, sodium salt, with methyloleate and sodium methoxide as a catalyst. ##STR5## The reactions iscarried out with an excess of methyl oleate as a solvent. To ensure thecompletion of the reaction, removal of side product methanol is carriedout.

Preparation of the HEPES oleate: sodium salt 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, sodium salt, is slowly added to astirring flask of methyl oleate in equal molar equivalents. The mixtureis stirred at room temperature for 15 minutes. Then a catalytic quantityof sodium methoxide is slowly added to the stirring mixture. Thereaction mix is stirred for less than five minutes. The reaction mix isthen slurried in acetone, then isolated and dried, affording greaterthan an 80% yield of the title oleate as a white to off-white solid, towit: ##STR6##

WORKING EXAMPLE I

In a 500 ml, 3 neck flask, 114 gms of methyl oleate is added with 100gms of HEPES, sodium salt (source: Angus Chemical); while stirring, themixed is warmed to 27-30° C. Once at 27-30° C., 0.4 gms of the sodiummethoxide catalyst is added. A mild exotherm is observed, and thecontents are slowly heated to 45-50° C. Once at 45-50° C., vacuum isapplied to remove methanol to ensure reaction completion.

After two hours at 45-50° C. and under vacuum, the reaction is complete.The vacuum is removed and acetone is added. With the reaction mix inacetone solution it is slowly cooled to 0-5° C. over two hours, andstirred for an additional six hours before isolating the HEPES oleate,sodium salt, in a filter funnel. The white to off-white solid product isthen placed in a vacuum oven at 50° C. at 28" of vacuum for six hours toremove any remaining acetone or methanol. A greater than 80% yield isobtained.

WORKING EXAMPLE 2

II. HEPES ether has the structure: ##STR7## HEPES, Na salt, is reactedwith ethylene oxide to form the ether of the below generic structuralformula: ##STR8##

HEPES Ether COMPOUND II WORKING EXAMPLE 3

III. HEPES urethane has the structure: ##STR9##

The reaction is carried out by adding isocyanate to the HEPES to formthe corresponding urethane below: ##STR10##

HEPES URETHANE COMPOUND III WORKING EXAMPLE 4

Certain other analogs and derivatives of HEPES can be synthesized, suchas Piperazine-N'-(2-ethane sulfonic acid) I. This can be seen as themono N-substituted analog of taurine. Another prospective analog is theN-Methylpiperazine-N'-(2 ethane sulfonic acid), II, that is readilyprepared. Also, the piperidine analog, III, of the piperazine ester(Comp I) may be usefully synthesized and evaluated, ##STR11##

WORKING EXAMPLE 5

PREPARATION OF HEPES-LINOLEATE ESTER-COMP V

The preparation of this ester is conducted by catalytically reactingHEPES, sodium salt, with methyl linoleate, CH₃ (CH₂)₄ HC: CH CH₂ --CH:CH(CH₂)₇ CO₂ CH₃, in equimolar amounts. The synthesis follows that ofworking Example I, yielding the ester V: ##STR12##

WORKING EXAMPLE 6

PREPARATION OF HEPES-ACETATE ESTER-COMPOUND VI

The preparation of this ester is conducted by catalytically reactingHEPES, sodium salt, with methyl acetate in equimolar amounts, yieldingthe Compound VI. The product is isolated and purified as described inWorking Example I. ##STR13## Protocol II for Studies with DermalIrritation

A new screening method was developed to make rapid assessments on theanti-inflammatory action of the new candidates. The procedure involvedthe use of a natural irritant, capsaicin*, which candidate is derivedfrom crushed hot peppers (capsicum), and measuring the amount oferythema reduction by the test candidates. Inflammatory reaction arecharacterzed by pain and tenderness associated with swelling andredness. Most of these clinical signs are attributed to the large influxof blood into the area. Vaso-constricting agents can reduce the amountof swelling and erythema. Hydrocortisone is, in fact, assayed by itsability to blanch the skin, which is a vasoconstrictive effect.

Applying a solution of 0.1% of capsaicin to the forearm of a humanvolunteer will produce an erythema in less than 20 minutes, usuallyunder five minutes. The degree of erythema is measured with an infra-reddetector. An infra-red beam of light at 818 nanometers is directed intothe skin over the test sie and the amount of infra-red returned to thesurface by reflection indicates the amount absorbed in the skin. Anuntreated skin area is used as a baseline control, a capsaicin treatedarea is used as the negative control, and a capsaicin+hydrocortisone (aknown vasoconstrictor) serves as the positive control. A positive effectmeans the erythema induced by the capsaicin is reduced, or iseliminated, as measured by the infra-red detector.

Results

The following reults were obtained with this evaluative protocol. Thecapsaicin was applied to the skin, and when erythema appeared 10-15minutes later, the test candidate agent was applied, and the opticalreflection measurements were made 30 minutes later.

    __________________________________________________________________________    Candidate Compound Tested                                                                              Millivolts (reflection voltage)                      __________________________________________________________________________    Hepes oleate (Compound I)                                                                              3.7                                                    Hepes linoleate (Compound V) 3.8                                              Hepes acetate (Compound VI) 3.0                                               Hydrocortisone 1% (positive control) 5.0                                      Baseline Control (no induced irritation and untreated skin) 3.5                                       Capsaicin only (negative control) 0.5               __________________________________________________________________________

The above data shows that the HEPES counteracted the erythema. Sinceerythema is a major part of the skin inflammation spectrum, reduction oferythema is a reliable indication of the potential biologicalanti-inflammatory action of a candidate agent. Indeed, each of thetested esters produced a 70% reduction in erythema compared to onepercent hydrocortisone ointment, an established anti-inflammatorysteroid.

What is claimed is:
 1. A prophylactic anti-inflammatory composition, indosage unit form, which consists essentially of:(a) one or morepharmacologically-acceptable carriers, and (b) an amount of a humanskin-compatible, organic derivative, being an ester, of a zwitterionicaminosulphonic acid of the formula. ##STR14## wherein M is an alkalimetal and R is a naturally occuring, straight, chain, saturated orunsaturated, aliphatic acid, which amount is effective to counteractdermal inflammation caused by skin irritants upon application to theaffected area, as measured by substantially reducing the degree oferythema, indicated by reduced swelling and redness.
 2. The compositionof claim 1 wherein the organic derivative is the oleic acid ester of4-(2-hydroethyl)-1-piperazine ethanesulfonic acid, having the molecularformula: ##STR15##
 3. The composition of claim 1 wherein the organicderiviative is the linoleic acid ester of4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, having themolecular formula:
 4. The composition of claim 1 wherein the organicderivative is the acetic acid ester of 4-(2-hydroxyethyl)-1-piperazineethanesulfanic acid having the molecular formula:
 5. A prophylacticanti-inflammatory composition, in dosage unit form, which consistsessentially of: (a) one or more pharmacologically-acceptable carriers,and,(b) an amount of a human skin-compatible, organic derivative, beingan ether, of a zwitterionic aminosulphonic acid of the formula.##STR16##
 6. A prophylactic anti-inflammatory composition, in dosageunit form, which consists essentially of: (a) one or morepharmacologically-acceptable carriers, and(b) an amount of a humanskin-compatible, organic derivative, being a urethane, of a zwitterionicaminosulphonic acid of the formula ##STR17## wherein R is an isocyanate(C₆ H₅ N CO) moiety.
 7. In a method of using an anti-inflammatorytopical composition containing one or more active ingredients fortreating dermal erythema, the improvement characterized by administeringa substantially curative amount of a formulated organic derivative,being an ester, of the molecular formula below: ##STR18## wherein M isan alkali metal and R is a naturally occuring, straight, chain,saturated or unsaturated, aliphatic acid, which amount is effective tocounteract dermal inflammation caused by skin irritants upon applicationto the affected area, as measured by substantially reducing the degreeof erythema, indicated by reduced swelling and redness.
 8. In a methodof using an anti-inflammatory topical composition containing one or moreactive ingredients for treating dermal erythema, the improvementcharacterized by administering a substantially curative amount of aformulated organic derivative of (HEPES) of the molecular formula below:##STR19## wherein M is an alkali metal and R is a naturally occuring,straight, chain, saturated or unsaturated, aliphatic acid, which amountis effective to counteract dermal inflammation caused by skin irritantsupon application to the affected area, as measured by substantiallyreducing the degree of erythema, indicated by reduced swelling andredness.
 9. In a method of using an anti-inflammatory topicalcomposition containing one or more active ingredients for treatingdermal erythema, the improvement characterized by administering asubstantially curative amount of an organic derivative of (HEPES) of themolecular formula below: ##STR20## wherein M is an alkali metal and R isa naturally occuring, straight, chain, saturated or unsaturated,aliphatic acid, which amount is effective to counteract dermalinflammation caused by skin irritants upon application to the affectedarea, as measured by substantially reducing the degree of erythema,indicated by reduced swelling and redness.